Pär Steneberg

Gpr40 Is Expressed in Enteroendocrine Cells and Mediates Free Fatty Acid Stimulation of Incretin Secretion

OBJECTIVE—The G-protein–coupled receptor Gpr40 is expressed in β-cells where it contributes to free fatty acid (FFA) enhancement of glucose-stimulated insulin secretion (1–4). However, other sites of Gpr40 expression, including the intestine, have been suggested. The transcription factor IPF1/PDX1 was recently shown to bind to an enhancer element within the 5′-flanking region of Gpr40 (5), implying that IPF1/PDX1 might regulate Gpr40 expression. Here, we addressed whether 1) Gpr40 is expressed in the intestine and 2) Ipf1/Pdx1 function is required for Gpr40 expression. RESEARCH DESIGN AND METHODS—In the present study, Gpr40 expression was monitored by X-gal staining using Gpr40 reporter mice and by in situ hybridization. Ipf1/Pdx1-null and β-cell specific mutants were used to investigate whether Ipf1/Pdx1 controls Gpr40 expression. Plasma insulin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and glucose levels in response to acute oral fat diet were determined in Gpr40 mutant and control mice. RESULTS—Here, we show that Gpr40 is expressed in endocrine cells of the gastrointestinal tract, including cells expressing the incretin hormones GLP-1 and GIP, and that Gpr40 mediates FFA-stimulated incretin secretion. We also show that Ipf1/Pdx1 is required for expression of Gpr40 in β-cells and endocrine cells of the anterior gastrointestinal tract. CONCLUSIONS—Together, our data provide evidence that Gpr40 modulates FFA-stimulated insulin secretion from β-cells not only directly but also indirectly via regulation of incretin secretion. Moreover, our data suggest a conserved role for Ipf1/Pdx1 and Gpr40 in FFA-mediated secretion of hormones that regulate glucose and overall energy homeostasis.

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop‐codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.

Dpp and Notch specify the fusion cell fate in the dorsal branches of the Drosophila trachea

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. We report that Dpp signaling is also required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling is Delta. In conditional Notch loss of function mutants additional tracheal cells adopt the fusion cell fate and ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch(ts1) mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches.

The Type 2 Diabetes–Associated Gene Ide Is Required for Insulin Secretion and Suppression of α-Synuclein Levels in β-Cells

Genome-wide association studies have identified several type 2 diabetes (T2D) risk loci linked to impaired β-cell function. The identity and function of the causal genes in these susceptibility loci remain, however, elusive. The HHEX/IDE T2D locus is associated with decreased insulin secretion in response to oral glucose stimulation in humans. Here we have assessed β-cell function in Ide knockout (KO) mice. We find that glucose-stimulated insulin secretion (GSIS) is decreased in Ide KO mice due to impaired replenishment of the releasable pool of granules and that the Ide gene is haploinsufficient. We also show that autophagic flux and microtubule content are reduced in β-cells of Ide KO mice. One important cellular role for IDE involves the neutralization of amyloidogenic proteins, and we find that α-synuclein and IDE levels are inversely correlated in β-cells of Ide KO mice and T2D patients. Moreover, we provide evidence that both gain and loss of function of α-synuclein in β-cells in vivo impair not only GSIS but also autophagy. Together, these data identify the Ide gene as a regulator of GSIS, suggest a molecular mechanism for β-cell degeneration as a consequence of Ide deficiency, and corroborate and extend a previously established important role for α-synuclein in β-cell function.

Hyperinsulinemia Enhances Hepatic Expression of the Fatty Acid Transporter Cd36 and Provokes Hepatosteatosis and Hepatic Insulin Resistance

Background: Enhanced hepatic expression of the fatty acid transporter Cd36 correlates with liver fat accumulation, hepatosteatosis, insulin resistance, and hyperinsulinemia. Results: Insulin increases hepatic Cd36 expression in a Pparγ-dependent manner. Conclusion: Hyperinsulinemia stimulates hepatic Cd36 expression, which correlates with the development of hepatosteatosis, hepatic insulin resistance, and dysglycemia. Significance: Hyperinsulinemia contributes to the development of hepatosteatosis and insulin resistance. Hepatosteatosis is associated with the development of both hepatic insulin resistance and Type 2 diabetes. Hepatic expression of Cd36, a fatty acid transporter, is enhanced in obese and diabetic murine models and human nonalcoholic fatty liver disease, and thus it correlates with hyperinsulinemia, steatosis, and insulin resistance. Here, we have explored the effect of hyperinsulinemia on hepatic Cd36 expression, development of hepatosteatosis, insulin resistance, and dysglycemia. A 3-week sucrose-enriched diet was sufficient to provoke hyperinsulinemia, hepatosteatosis, hepatic insulin resistance, and dysglycemia in CBA/J mice. The development of hepatic steatosis and insulin resistance in CBA/J mice on a sucrose-enriched diet was paralleled by increased hepatic expression of the transcription factor Pparγ and its target gene Cd36 whereas that of genes implicated in lipogenesis, fatty acid oxidation, and VLDL secretion was unaltered. Additionally, we demonstrate that insulin, in a Pparγ-dependent manner, is sufficient to directly increase Cd36 expression in perfused livers and isolated hepatocytes. Mouse strains that display low insulin levels, i.e. C57BL6/J, and/or lack hepatic Pparγ, i.e. C3H/HeN, do not develop hepatic steatosis, insulin resistance, or dysglycemia on a sucrose-enriched diet, suggesting that elevated insulin levels, via enhanced CD36 expression, provoke fatty liver development that in turn leads to hepatic insulin resistance and dysglycemia. Thus, our data provide evidence for a direct role for hyperinsulinemia in stimulating hepatic Cd36 expression and thus the development of hepatosteatosis, hepatic insulin resistance, and dysglycemia.

Insulin-degrading enzyme prevents α-synuclein fibril formation in a nonproteolytical manner.

The insulin-degrading enzyme (IDE) degrades amyloidogenic proteins such as Amyloid β (Αβ) and Islet Amyloid Polypeptide (IAPP), i.e. peptides associated with Alzheimer’s disease and type 2 diabetes, respectively. In addition to the protease activity normally associated with IDE function an additional activity involving the formation of stable, irreversible complexes with both Αβ and α-synuclein, an amyloidogenic protein involved in Parkinson’s disease, was recently proposed. Here, we have investigated the functional consequences of IDE-α-synuclein interactions in vitro. We demonstrate that IDE in a nonproteolytic manner and at sub-stoichiometric ratios efficiently inhibits α-synuclein fibril formation by binding to α-synuclein oligomers making them inert to amyloid formation. Moreover, we show that, within a defined range of α-synuclein concentrations, interaction with α-synuclein oligomers increases IDE’s proteolytic activity on a fluorogenic substrate. We propose that the outcomes of IDE-α-synuclein interactions, i.e. protection against α-synuclein amyloid formation and stimulated IDE protease activity, may be protective in vivo.

PAN-AMPK activator O304 improves glucose homeostasis and microvascular perfusion in mice and type 2 diabetes patients

AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which - in diet-induced obese mice - increased glucose uptake in skeletal muscle, reduced β cell stress, and promoted β cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.

The 17,18-epoxyeicosatetraenoic acid–G protein–coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques

Background: Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18‐Epoxyeicosatetraenoic acid (17,18‐EpETE) is a recently identified new class of antiallergic and anti‐inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. Objective: We evaluated the effectiveness of 17,18‐EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18‐EpETE. Methods: Contact hypersensitivity was induced by topical application of 2,4‐dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT‐PCR analyses. Neutrophil mobility was examined by means of imaging analysis in vivo and neutrophil culture in vitro. The receptor for 17,18‐EpETE was identified by using the TGF‐&agr; shedding assay, and the receptors involvement in the anti‐inflammatory effects of 17,18‐EpETE was examined by using KO mice and specific inhibitor treatment. Results: We found that preventive or therapeutic treatment with 17,18‐EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18‐EpETE was recognized by G protein–coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant‐induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18‐EpETE was abolished in the absence or inhibition of GPR40. Conclusion: 17,18‐EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases. Graphical abstract: Figure. No caption available.